BACTERIOLOGY LECTURE
S.Y. ‘23 - ‘24 | BY HUEN MINGARINE
MODULE
1.0 Staphylococcus species
● classified the family Staphylococcaceae
● Historically, the genus Staphylococcus was
included in the family Micrococcaceae which
also includes the Genus Micrococcus
● Several of the Micrococcus species are now
reclassified into 4 other genera. These
genera have now reorganized into 2 families
Micrococcaceae, and Dermococcaceae
● There are, currently several species and
subspecies (45 species and 21 subspecies)
within the genus Staphylococcus
● The coagulase-positive Staphylococcus
aureus, two coagulase-negative species, S.
epidermidis and S. saprophyticus are seen
frequently in human infections
● S. hyicus – causes an infectious dermatitis
in swine
● S. chromogenes (formerly a subspecies of S.
hyicus) – causing infection in swine, cattle,
and goat
● S. intermedius – has been isolated from
several types of infections in dogs
● S. delphini – dolphins
● S. felis – domesticated cats
● S. schleiferi subsp. Coagulans – dogs
● S. lutrae – sea otters
● S. schleiferi subsp coagulans – cause
external otitis in dogs
● S. pseudointermedius – in dogs, cats, horse,
parrots
1.1 General Characteristics
● Gram-positive cocci, 0.5 - 1.5 um in diameter,
and typically in grape-like clusters.
● Non-motile
● Non–spore-forming
● Facultative anaerobes
● Grow most rapidly at 37°C
● Halotolerant
● Catalase(+)
● Fermentative
● Nitrate reduction(+)
● Coagulase(+)
● DNase(+)
● Mannitol fermenter
1.2 Habitat and Transmission
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● Habitat is the human nose and skin
- S. aureus are found in the anterior
nares of 20% to 40% of adults,
carried by healthy individuals
intermittently rather than
chronically.
- Those on the skin are believed to be
contaminants from the nose.
- Other sites of colonization include
the nasopharynx, perineum, the
axillae, and the vagina.
● Transmission is via contaminated hands
- Staphylococcal infections are most
commonly acquired endogenously
from colonized anterior nares or by
direct contact with someone
carrying S. aureus.
- From the nasal site, the bacteria are
shed to the exposed skin and
clothing of the carrier and others
with whom they are in direct
contact.
- Spread is augmented by touching
the face and, of course, nose
picking.
1.3 Pathogenesis and Clinical
Manifestations
● Although this organism is frequently a part
of the normal human microflora, it can cause
significant opportunistic infections under the
appropriate conditions.
● Factors which may predispose an individual
to serious S. aureus infections include:
1. breaks in the continuity and
integrity of mucosal and cutaneous
surfaces
2. presence of foreign bodies or
implants (e.g., sutures, intravenous
lines, prosthetic devices)
3. prior infection with other agents,
particularly viruses (e.g., influenza)
4. underlying diseases with defects in
cellular or humoral immunity, either
congenital or acquired, (e.g.,
defects in leukocyte chemotaxis,
defects in opsonization by
antibodies,
hypogammaglobulinemia or
complement component
deficiencies and/or defects)
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5. chronic underlying diseases such
as malignancy, alcoholism, and
heart disease
6. therapeutic or prophylactic
antimicrobial administration
2.0 Diseases
2.1 Cutaneous diseases
● Staphylococcal infections of the skin
(through wounds, follicles, or skin glands)
are pyogenic (pus-forming), and often
presents as an inflamed, fibrous lesion
enclosing a core of pus called an abscess
Folliculitis – mild inflammation of the
superficial dermis that is restricted
to ostia (opening) of the hair
follicles and is characterized by the
presence of small, reddish, painful
lesions and the absence of
systemic symptoms
– Stye – eyelid
Hidradenitis – chronic or relapsing inflammatory
suppurativa disease of the skin, involving the
apocrine gland-bearing areas
axillae, groin, perineal (perineum —
an area between the thighs) and
perianal regions
– characterized by presence of
multiple lesions associated with
blocked and infected apocrine
sweat glands
– While local pain, swelling and
erythema are present, systemic
symptoms such as fever is usually
absent
Furuncle or – a deeper-seated infection of the
boil hair follicles
– results when the inflammation of
single hair follicle or sebaceous
gland progresses into a large, red,
extremely tender abscess or pustule
– Furuncles often appear in clusters
(known as furunculosis) in parts of
the body such as the buttock,
breasts, axillae, and back of the
neck where skin rubs against other
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skin or clothing
Carbuncle – a larger and deeper lesion created
by aggregation and interconnection
of a cluster of furuncles
– extremely painful and can even be
fatal in elderly patients when they
give rise to systemic disease
– usually found in areas of which
tough skin such as on the back of
the neck
Impetigo – a staphylococcal skin infection
that is not confined to follicles and
skin glands
– characterized by bubble-like
epidermal sweeping that can break
and peel away like a localized form
of scalded skin syndrome
– usually present on exposed areas,
especially the face, and may spread
to surrounding areas by
autoinoculation (by the infected
person himself, to other sites of his
body)
Non-bullous impetigo
– begins as a single red macule
(patch) or papule that quickly
becomes a vesicle.
– The vesicle rupture and forms an
erosion, and the lesion and its
contents dry to form a
characteristic honey-colored crusts
with erythematous (reddish)
margins that may be pruritic (itchy).
Bullous impetigo
– is a localized form of
staphylococcal scalded skin
syndrome that commonly affects
neonates but can also occur in older
children and adults.
– Superficial vesicles progress to
enlarging bullae (singular; bulla
plural; vesicle or blister) with sharp
margins with no surrounding
erythema. When the bullae ruptures,
yellow crust with oozing results.
– Bullous impetigo is usually found
in moist, intertriginous areas (e.g.,
the diaper area, axillae, neck folds).
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2.2 Toxigenic diseases
● Toxigenic disease caused by S. aureus can
present itself as a toxemia due to the
production of toxins in the body or as food
intoxication, the ingestion of preformed
toxin.
1. Staphylococcal food poisoning
- a gastrointestinal illness caused by
eating foods contaminated with
toxins produced by the S. aureus,
thus it is a food intoxication
- characterized by acute symptoms
that appear in 2 to 6 hours after
ingestion of the toxin-contaminated
food
- Emesis (vomiting) is often
projectile, and diarrhea is less
frequent
- Recovery is rapid, usually within 24
hours
- associated with eating foods such
as custards, sauces, cream
pastries, processed meats, chicken
salad, or ham that have been
contaminated by handling and then
left unrefrigerated for a few hours
- Because of the high salt tolerance
of S. aureus, even foods that
contain salt as a food preservative
are not exempt
- The toxins produced by the
multiplying bacteria does not alter
the food’s taste or smell
2. Staphylococcal Scalded Skin Syndrome
(SSSS) (AKA Ritter's disease)
- usually seen in neonates (newborns
or children less than a month old)
and infants less than 5 years
- characterized by widespread
erythema and the appearance of
bullous lesions over large areas of
the body, with subsequent
sloughing of the superficial skin
layers
- exposure of large areas of d e n u d
e d (stripped of surface layers) and
raw skin
- most cases, desquamation occurs
and symptoms wane over 5-7 days
3. Staphylococcal toxic shock syndrome (TSS)
- initially a flu-like illness
characterized by fever, hypotension,
and rash on the skin that resembles
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a sunburn, and can involve any
region of the body, including the
lips, mouth, eyes, palms and soles
- patients who survive the initial
phase of the infection, the rash is
followed by desquamation 1 – 2
weeks after onset
- involvement of multiple (three or
more) organ systems with varying
symptoms of vomiting, diarrhea,
renal failure, headache, chills, sore
throat and conjunctivitis
- Initially, the disease was noted
most frequently in women, with
onset mainly occurring during
menstruation; association between
the onset of disease and the use
high-absorbency tampons during
menses
- non-menstrual-associated TSS was
reported in males and females as
complication of staphylococcal
abscesses or systemic infections
2.3 Miscellaneous Systemic Infections
● Most systemic staphylococcal infections
have a focal pattern, spreading from a local
cutaneous infection to other sites. These
include:
- osteomyelitis (bone infection)
- pneumonia which usually occurs
after influenza viral infection
- bacteremia with consequences
such as, but not limited to,
endocarditis, arthritis, and
meningitis
3.0 Virulence Factors
● S. aureus expresses many potential virulence
factors that promote colonization of, spread
in, or damage to host tissues, or enhance
their survival within the host.
● These include a variety of cellular structures
and extracellular substances:
Microcapsule – consists of polysaccharides
– can be visualized only by
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electron microscopy (hyaluronic acid) in tissue, thus

– mediates attachment to host spreads the organism to
cells or tissues, and inhibits adjacent areas in tissues
phagocytosis by
polymorphonuclear leukocytes Lipase – hydrolyzes lipids and help
unless specific antibodies are the spread of the organism in
present cutaneous and subcutaneous
– At least 11 serotypes have tissues
been identified, with types 5
and 8 responsible for the Staphylokinase – plasminogen activator. This
majority of infections factor dissolves fibrin clot (a
fibrinolysin)
Protein A – a bacterial surface protein in – Localized fibrinolysis might
some S. aureus strains aid in the spread of infection to
– binds to the Fc portion of IgG, contiguous tissues
which, therefore, will no longer
be capable of binding to Fc Phosphatidylino – affects tissues of the host
receptor on phagocytes, thus sitol-specific making them more susceptible
preventing opsonization phospholipase C to damage and destruction by
– blocks complement fixation complement components and
– S. aureus with Protein A products during complement
bound to the Fc portion of IgG activation
molecules are used as reagents
for identification of certain β-lactamase – under plasmid-control
organisms transmitted by transduction
and perhaps also by
Catalase – inactivates toxic hydrogen conjugation and provides
peroxide and free radicals resistance to β-lactam
formed by the myeloperoxidase antibiotics such as penicillin,
system within the phagocytic penicillin derivatives (penams),
cells, which are the primary and cephalosporins
killing mechanisms within the – These antibiotics all have a
phagolysosome common element in their
molecular structure: a
Coagulase – promotes conversion of four-atom ring known as a
fibrinogen to fibrin causing β-lactam
plasma to clot. Fibrin coat the – Through hydrolysis, the
bacterial cells, hiding their β-lactamase breaks the
antigenic surface, thus β-lactam ring open,
protecting them from deactivating the molecule's
phagocytosis and immune antibacterial properties
response
Toxic Shock – a superantigen
Deoxyribonuclea – hydrolyzes DNA Syndrome Toxin – promotes the manifestations
se (DNase) – facilitates the spread of (TSST-1) of toxic shock syndrome. The
bacteria by liquefying and gene for TSST-1 is found in
decreasing the viscosity of about 20% of S. aureus isolates
abscess materials – Superantigens are protein
toxins that stimulate T cells
Hyaluronidase – also known as the non-specifically without normal
“spreading factor” antigenic recognition
– hydrolyzes the intercellular
matrix of acid Exfoliative – epidermolytic toxins that
mucopolysaccharides toxins (or facilitate dissolution of the

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BACTERIOLOGY LECTURE
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exfoliatins) mucopolysaccharide matrix of
the stratum granulosum in the
epidermis resulting in the
intraepithelial splitting of
cellular linkages seen in
Staphyloccoccal Scalded Skin
Syndrome (SSSS).
– There are two distinct
proteins of the same molecular
weight:
a. Exfoliative toxin A
(ET-A) is produced by
lysogenized strains of
S. aureus
b. Exfoliative toxin B
(ET-B) is
plasmid-mediated.
Both have
superantigen
activities.
Enterotoxins A – heatstable exotoxins which
through E, G-J, are responsible for the clinical
K-R and U, V features of staphylococcal
food poisoning
– Heating the food may not
prevent the disease because
inactivation of staphylococcal
enterotoxin requires 100oC for
at least 30 minutes
– S. aureus enterotoxins are
superantigens. Approximately
50% of S aureus strains can
produce one or more of them
NOTE
MRSA are strains of the S. aureus that are resistant
to the action of methicillin, and related beta-lactam
antibiotics (e.g., penicillin, oxacillin, amoxicillin).
MRSA has evolved resistance not only to
beta-lactam antibiotics, but to several classes of
antibiotics.
Resistance to methicillin is due to the presence of
an altered penicillin-binding protein called PBP2a
(or PBP2′) that results from acquisition of a
chromosomal gene called mecA which is located
within the large mobile staphylococcal
chromosomal element known as the SCCmec
(staphylococcal cassette chromosome).
Most MRSA infections occur in people who've been
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in hospitals or other health care settings, such as
nursing homes and dialysis centers. When it occurs
in these settings, it's known as health
care-associated MRSA (HA-MRSA). HAMRSA
infections usually are associated with invasive
procedures or devices, such as surgeries,
intravenous tubing or artificial joints. HA-MRSA can
spread by health care workers touching people with
unclean hands or people touching unclean
surfaces. Another type of MRSA infection has
occurred in the wider community — among healthy
people. This form, community-associated MRSA
(CA-MRSA), often begins as a painful skin boil. It's
usually spread by skin-toskin contact.
Blood Cell Toxins (Hemolysins and Leukocidins)
– hemolysins lyse red blood cells by disrupting
their cell membranes
– Leukocidins damage cell membranes of
neutrophils and macrophages, causing them to lyse
– help incapacitate the host’s phagocytic line of
defense
⍺ (alpha)-Hemolysin
– heterogeneous protein that facilitates formation
of pores on the target eukaryotic cell membrane
through which monovalent cations can pass,
leading to osmotic swelling and rupture of the cell.
– This potent toxin lyses red blood cells of various
mammals and damages monocytes, macrophages,
lymphocytes, skeletal muscle, heart, and renal
tissue as well.
– It is also active against thrombocytes (platelets)
and may contribute to septic thrombotic events
during S. aureus bacteremia.
β (beta)-Hemolysin
– a sphingomyelinase that degrades sphingomyelin
in the cell membrane and therefore is toxic for
many kinds of cells. It is cytotoxic not only to
human erythrocytes but also to monocytes.
– β-hemolysin is said to be inactive against
granulocytes, lymphocytes, and fibroblasts. It is a
protein exotoxin described as a “hot-cold”
hemolysin; its hemolytic properties are enhanced
by subsequent exposure to red blood cells to low
temperatures. A lysogenic bacteriophage is known
to encode the toxin.
δ (delta)-Hemolysin
– is a small peptide that acts primarily as a
surfactant or a detergent-like molecule that
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interacts with cell membranes and forms channels 5.0 Laboratory Diagnosis
resulting in slow leakage of cellular contents. It
may have a role in S. aureus diarrheal diseases.
5.1 Staphylococcus epidermidis
γ (gamma)-Hemolysin
– a leukocidin that lyses white blood cells and is
composed of two proteins designated S and F. ● Gram-positive cocci, 0.5 - 1.5 um in diameter,
– It is capable of efficiently lysing white blood cells and typically in grape-like clusters.
by causing pore formation in the cellular ● Non-motile
membranes that increase cation permeability. ● Non–spore-forming
– Affected cells undergo degranulation of the ● Facultative anaerobes
cytoplasm, cell swelling, and lysis. This leads to ● Grow most rapidly at 37°C
massive release of inflammatory mediators which ● Halotolerant
are responsible for necrosis and severe ● Catalase(+)
inflammation. ● Fermentative
● Nitrate reduction(+)
Panton-Valentine Leukocidin (PVL) ● Coagulase(-)
– is produced by lysogenized strains of S. aureus. ● DNase(-)
This pore-forming toxin is active against ● Non-mannitol fermenter
neutrophils and causes tissue necrosis. It consists ● most commonly encountered staphylococcal
of two components designated as S and F that act species
synergistically on the white blood cell membrane as ● Comprises 99% of the normal flora of the
described for γ toxin. skin
● Considered opportunistic pathogens, but are
substantially less virulent than S. aureus
4.0 Prevention and Control ● Associated with nosocomial infections
related to use of contaminated medical
devices
● Infection and spread thereof may be - Endocarditis - associated with
prevented by cleanliness, hygiene, and prosthetic heart valves; also
aseptic management of lesions. pacemaker wires, implanted
● Application of topical antiseptics such as defibrillators, and vascular grafts
mupirocin to nasal or perineal carriage sites - Arthritis - associated with
may diminish shedding of dangerous prosthetic joint, or hip implant
organisms. - Urinary tract infection (UTI) —
● In health care facilities, surveillance of associated with indwelling urinary
anterior nares colonization among staff in catheters
health facilities particularly those who man - Bacteremia - associated with IV
the Intensive Care Unit by periodic swabbing. catheters
● Health care workers are advised to strictly ● Pathogenesis is related to production of
adhere to infection control policies by adherent slime resulting in biofilm formation
wearing gloves and washing hands before on the surface of a prosthetic device
and after patient contact.
● Clothes and bedding that may cause
reinfection should be dry-cleaned or washed 5.2 Staphylococcus saprophyticus
at a sufficiently high temperature (70°C or
higher) to destroy staphylococci. ● Gram-positive cocci, 0.5 - 1.5 um in diameter,
● Chemoprophylaxis is effective in surgical and typically in grape-like clusters.
procedures such as hip and cardiac valve ● Non-motile
replacements, in which infection with ● Non–spore-forming
staphylococci can have devastating ● Facultative anaerobes
consequences. ● Grow most rapidly at 37°C
● Halotolerant
● Catalase(+)

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● Fermentative
● Nitrate reduction(+)
● Coagulase(-)
● DNase(-)
● Urease (+)
● Mannitol fermentation (variable)
● Primarily a saprophyte
● Gastrointestinal tract (primarily the rectum)
as the primary site of colonization in
humans; also part of the normal flora of
female genitourinary tract (urethra, cervix),
perineum
● Second most common cause of
community-acquired UTI in young, sexually
active females (most common is Escherichia
coli)
● Pathogenesis is related to production of
adherent slime and urease (an enzyme that
hydrolyzes urea in urine resulting in the
formation of ammonia and ammonium
carbonate, making the urine alkaline and
favoring bacterial growth)
6.0 Laboratory Diagnosis of
Staphylococcus species
6.1 Specimens
● Pus from wound and other pyogenic
infections
● Blood from septicemia
● CSF from meningitis
● Sputum from respiratory infections
● Urine from UTI
● Nasal swab from suspected carriers
6.2 Procedure Notes
A. Microscopy
- G+ cocci, 0.5-1.5 µm in diameter,
and grape-like clusters
- A direct smear, one that is prepared
from a clinical material such as pus
or CSF, usually reveals G+ cocci
arranged as single cells, pairs,
tetrads, and short chains.
- The direct smears should also be
examined for the presence of
inflammatory cells. These cells,
background tissue debris and
proteinaceous material generally
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stain pink to red in properly gram
stained specimens.
B. Cultural method
- Staphylococcus species grow well
on routine laboratory media:
Blood – Enriched medium that supports the
agar growth of staphylococci and permits
medium observation of the pattern of
(BAM) hemolysis of blood
Mannitol – MSA is both a selective and
salt agar differential medium for staphylococci.
(MSA) Uninoculated MSA appears light to
medium rose red in color
– The high salt (7.5% NaCl)
concentration inhibits the growth of
other organisms (except for
enterococci), and selectively allows
the growth of halotolerant
staphylococci
– Mannitol fermentation results in
acids indicated by a change in the
phenol red indicator to yellow, and
aids in the differentiation of
staphylococcal species
Columbia – Sheep blood supports the growth of
CNA Agar fastidious bacteria and allows
detection of hemolytic reactions.
– Antimicrobial agents, colistin and
nalidixic acid, render the medium
selective for G+ bacteria by inhibiting
G- bacteria.
Phenyleth – Sheep blood supports the growth of
yl Alcohol fastidious bacteria particularly G+
(PEA) cocci.
Agar – The medium should not be used for
determination of hemolytic reactions
since atypical reactions may be
observed.
– PEA inhibits G- bacteria.
Vogel-Jo – Selectivity of the medium is
hnson achieved by potassium tellurite,
(VJ) Agar lithium chloride and glycine which
inhibit nonstaphylococcal organisms,
both G+ and G- bacteria.
– Tellurite is reduced to metallic
tellurium by S. aureus producing
colonies that are black or gray-black in
color; mannitol is degraded into acid
which is indicated by phenol red,
which changes the color of the
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medium to yellow.
– Small, gray-black, not surrounded by
yellow zones (may become deeper red
in color) presumptively identifies S.
epidermidis
Chapman – It is selective due to relatively high
Stone salt content, although it is reduced to
Agar 5.5%.
– Gelatin serves as a substrate for
gelatinase activity; ammonium sulfate
allows detection of gelatin hydrolysis
which is characterized by clear zone
around the colonies.
– differential due to mannitol
fermentation. To determine mannitol,
add few drops of bromcresol purple to
areas on the medium; yellow color
indicates mannitol fermentation
– Yellow colonies surrounded by clear
halo presumptively identifies S. aureus
– White nonpigmented colonies with
or without clear zone probably is S.
epidermidis
Baird-Par – Tellurite, lithium chloride and glycine
ker Agar have inhibitory action for organisms
other than staphylococci
– Sodium pyruvate is incorporated in
order to stimulate the growth of S.
aureus without destroying the
selectivity
– Reduction of tellurite to tellurium
produces a black coloration
– Addition of egg yolk is used to
demonstrate proteolysis and lipolysis
by lecithinase and lipase respectively
producing clear zones around the
colonies
– S. aureus: Black, shiny, convex
colonies 1-5 mm in diameter with a
narrow, white edge.
– S. epidermidis: Black, shiny, irregular
shape. Opaque zone develop around
the colonies after 24 hours.
● Interpretation of colonial morphologies is
usually performed after 24 to 48 hours of
incubation at 35-37 oC
● Colonial characterization:
Blood – Staphylococcal colonies (a) are
agar medium to large (1-2 mm in diameter),
medium off-white or gray, smooth, entire,
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(BAM) slightly raised, low convex, opaque,
and butyrous in consistency
– Colonies of some S. aureus are
usually large (4-6 mm in diameter).
– Some strains (b) produce golden
yellow pigments "lipochrome" so
appear creamy yellow or
yellow-orange (hence, the name
"aureus", meaning "golden")
– Others (c) may have a distinct or
hazy zone of β-hemolysis around the
colonies; this hemolytic property may
become apparent only after prolonged
incubation
– S. epidermidis produce small to
medium, opaque, gray-white colonies;
most colonies nonhemolytic;
slime-producing strains are extremely
sticky and adhere to the agar surface
– S. saprophyticus colonies are large,
entire, very glossy, smooth, opaque,
butyrous, convex; usually white but
colonies can be yellow or orange
Mannitol – Mannitol-fermenting staphylococci
salt agar (a) produce growth of yellow colonies
(MSA) with yellow discoloration of the
medium. This is typical of S. aureus.
However, other staphylococci
(particularly S. saprophyticus) may
also ferment mannitol and thus
resemble S. aureus on this medium
– Non-mannitol fermentation (b) by S.
epidermidis and several other species
produce small red colonies with no
color change to the medium
7.0 Identification Tests
7.1 Catalase Test
● When performed with bacterial isolates that
are G+ cocci, this test distinguishes
staphylococci which are strongly
catalase-positive from streptococci
(catalase-negative)
● Based on the ability of the bacteria to p r o d
u c e t hecatalaseenzymethat
decomposes hydrogen peroxide into water
and oxygen. The evolution of oxygen results
in effervescence (i.e.,rapid bubble formation)
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7.2 Coagulase test
● single most reliable characteristic for
identifying S. aureus The conventional
coagulase test may be performed by the
slide or tube procedures
● The coagulase enzyme produced by S.
aureus binds plasma fibrinogen and
activates a cascade of reactions causing
plasma to clot.
a. Slide coagulase test
- screening test for S. aureus. Strains
that are negative with the slide
coagulase test must be confirmed
with a tube coagulase test
- Based on the presence of bound
coagulase or “clumping factor” on
the surface of the cell wall of most
strains of S. aureus. This factor
reacts directly with and converts
fibrinogen in plasma into fibrin,
causing rapid cell agglutination
- Some human coagulase-negative
species (e.g., S. lugdunensis and S.
schleiferi subsp schleiferi) produce
clumping factor and may be slide
coagulase-positive
- The test can be performed with
growth from blood agar, or other
nonselective medium, but should
not be performed from media
having a high salt content (e.g.,
mannitol salts agar) since the high
salt causes some strains of S.
aureus to autoagglutinate.
b. Tube coagulase test
- confirmatory test for S. aureus
since free coagulase is secreted by
virtually all strains of S. aureus
- Based on the detection of free
coagulase which is secreted
extracellularly by S. aureus. The free
coagulase reacts with a substance
in the plasma called
coagulase-reacting factor (CRF),
which in turn reacts with fibrinogen
in plasma to form fibrin (clot
formation).
- Some animal isolates (S.
intermedius, S. hyicus, S. delphini,
S. schleiferi subsp. coagulans and
strain of S. schleiferi subsp.
schleiferi ) may be tube
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coagulase-positive. Other
phenotypic tests distinguish them
from S. aureus.
- When observing for any degree of
clot formation (partial or complete)
after incubation, gently tilt the tube;
do NOT agitate as a small clot
formed may be dislodged and will
not reform anymore.
- Tube coagulase tests that are
negative after 4 hours incubation at
35°C should be held at room
temperature and read again after 18
to 24 hours, because some strains
will produce fibrinolysin
(staphylokinase) on prolonged
incubation at 35°C, causing
dissolution of the clot during the
incubation period.
NOTE
– The medium for both slide coagulase and tube
coagulase procedures is rabbit plasma with EDTA,
which is commercially available in lyophilized form
– Rabbit plasma has high amounts of
coagulase-reacting factor (CRF); and, EDTA is the
preferred anticoagulant.
– Human plasma must not be used because it
contains variable amounts of CRF and may contain
anti-staphylococcal antibodies.
– Citrated plasma should not be used as some
bacteria that are able to utilize citrate (such as
enterococci) will yield positive results if they are
mistaken for staphylococci; always perform a
catalase test first.
7.3 Novobiocin test
● one of the methods to distinguish between
coagulase-negative staphylococci, S.
epidermidis and S. saprophyticus
● S. epidermidis as an agent involved in
several infectious processes and the
recognized clinical significance of S.
saprophyticus in urinary tract infections
● performed as a disk susceptibility test using
a novobiocin disk (NB,5 µg)
● some human staphylococcal species (S.
cohnii subspecies, S. hominis subsp.
novobiosepticus, S. xylosus, some S.
pseudolugdunensis) are resistant to
novobiocin
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7.4 Deoxyribonuclease (DNase) test
● DNase test detects bacterial
deoxyribonuclease which is an extracellular
enzyme that depolymerizes (breaks down)
DNA into subunits composed of nucleotides
● Inoculate, either by streak inoculation or spot
inoculation, a DNase test agar plate
(containing deoxyribonucleic acid), and
incubate at 35 oC for 24-48 h; after
incubation, flood with 1N hydrochloric acid
(HCl) which will precipitate DNA.
● A clear area surrounding the growth after
addition of 1N HCL indicates a positive
reaction for DNase activity. A negative
reaction is indicated by no clearing and a
cloudy precipitate around the colonies.
● Alternative method: The test may also be
carried out in agar plates with dye, such a s
m e t h y l g r e e n a n d toluidine blue. This
eliminates the need to add 1N HCl.
7.5 Thermostable Endonuclease Test
● The same DNase test agar is used; 3-mm
holes are cut into the agar with a sterile cork
borer, and the wells are filled with a 24-hour
broth culture of the test organism that has
been boiled in a water bath for 15 minutes.
The plate is incubated overnight at 35°C.
● S. aureus which is thermostable
endonuclease positive will show a pink zone
surrounding the well containing the boiled
suspension. Negative test is when the blue
color in the medium remains
8.0 Micrococcus species
● often resemble and ,therefore, are easily
confused with staphylococci
● Most species are found free-living in the
environment including air, soil, various
extreme environments (e.g., marine
sediment, deep sea mud, activated sludge),
and food (usually fermented seafood).
● In humans, Micrococcus species are
considered part of the normal microbiota of
the skin, mucosa, and oropharynx. Their
mode of transmission is still uncertain, and
BSMLS 2 | 2ND SEMESTER | MIDTERM | BACTERIOLOGY LECTURE | BY HUEN MINGARINE
MODULE 4
they are rarely implicated in human
infections. When infections occur,
particularly in hosts with compromised
immune systems, such as HIV patients, they
likely involve endogenous strains (those that
are part of the host’s normal flora).
● rarely clinically significant
● typically appear as gram-positive cocci in
tetrads, rather than in clusters and usually
larger (0.5 -3.5 µm) than staphylococci
● Some micrococci are pigmented bacteria
- M. luteus produces yellow colonies
and M. roseus produces reddish
colonies
- The yellow colonies of M. luteus
may be mistaken for the pigmented
colonies of S. aureus.
9.0 Methods
9.1 Oxidation-Fermentation (O-F) test
● O-F test determines whether bacteria can
utilize glucose in the presence or absence of
oxygen.
● A semisolid medium containing glucose as
the sole carbohydrate in the medium and
bromothymol blue as pH indicator is used.
● Two tubes are inoculated by stabbing; one
tube (designated as the "closed tube") is
overlaid with sterile mineral oil which shields
it from oxygen in air, while the other tube
("open tube") is exposed to oxygen.
● Micrococci are oxidative because they are
aerobic bacteria; they produce acids from
glucose only in the presence of oxygen, not
in its absence. Medium in the open tube
turns yellow but not in the closed tube.
● Staphylococci are fermentative, utilizing
glucose, and produce acids in the presence
of oxygen as well as in its absence. This is
related to the fact that staphylococci are
facultative anaerobes. This is evidenced by
C. bound coagulase by yellow color change
of the medium in both open and closed
tubes.
9.2 Susceptibility to Bacitracin, 0.04 U
and Furazolidone, 100 µg
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BACTERIOLOGY LECTURE
S.Y. ‘23 - ‘24 | BY HUEN MINGARINE MODULE 4

● This test is performed as an agar disk

susceptibility procedure using commercially 9.5 Production of acids from glycerol
available disks: in the presence of erythromycin
- Taxo A disk contains 0.04 U
bacitracin
● The test uses a medium containing glycerol
- FX disk contains 100 µg
(1%) and erythromycin (0.4 µg/mL). It is
furazolidone
prepared with an enriched agar base
● Micrococci are susceptible to bacitracin
containing bromocresol purple indicator and
producing zones of 10 m m o r g r e a t e r, b
poured into Petri plates. Several colonies of
u t a r e furazolidone-resistant and shows
the isolate are streaked as a single line on
zones of inhibition up to 9 mm.
the medium and the plate is incubated for up
● Staphylococci are bacitracin-resistant and
to 3 days at 35°C.
grow to the edge of the Taxo A disk; but,
● Micrococci will not produce acids from
furazolidone-susceptible, thus are inhibited
glycerol, so the medium will remain purple in
by furazolidone and show zones of 15 mm or
color.
more.
● Staphylococci will produce acids from
glycerol causing a yellow color change in the
9.3 Lysostaphin test medium.

● Lysostaphin is an endopeptidase enzyme

that can lyze the cell wall of staphylococci
but not micrococci.
● Lysostaphin solution is added to a bacterial
suspension and incubated at 35-37 oC for 2
hours.
● Micrococci are lysostaphin resistant —
bacterial suspension remains turbid; it does
not clear.
● Staphylococci are lysostaphinsusceptible —
there is clearing of the bacterial suspension.

9.4 Modified oxidase test

● Modified oxidase test is also known as the

microdase test; it is recommended for
gram-positive, catalase-positive cocci only.
● uses filter paper disks impregnated with
tetramethyl-p phenylenediamine
dihydrochloride in dimethyl sulfoxide
(DMSO). A colony from the growth medium
is removed with an applicator stick and
rubbed onto the disk
● Micrococcus species are modified
oxidase-positive; the test is indicated by
development of a blue-purple color within 30
seconds.
● Staphylococcus species are generally
modified oxidase-negative (no change in
color).

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